1. Field of the Invention
The present invention relates generally to immunological assay techniques and specifically to a method for identifying an antibody that binds to a novel epitope through the use of solid-phase capture reagents that bind antigen at a preselected epitope and panning of a combinatorial library.
2. Description of Related Art
Generally, any preparation of polyclonal or monoclonal antibodies can be used as a source of antibody molecules for cloning purposes. The use of filamentous phage display vectors, referred to as phagemids, has been repeatedly shown to allow the efficient preparation of large libraries of monoclonal antibodies having diverse and novel immunospecificites. The technology uses a filamentous phage coat protein membrane anchor domain as a means for linking gene-product and gene during the assembly of filamentous phage replication, and has been used for the cloning and expression of antibodies from combinatorial libraries (Kang, et al, Proc. Natl. Acad. Sci., U.S.A., 88:4363, 1991). Combinatorial libraries of antibodies have been produced using both the cpVIII membrane anchor and the cpIII membrane anchor (Barbas, et al, Proc. Natl. Acad. Sci., U.S.A., 88:7978, 1991).
The diversity of a phagemid library can be manipulated to increase and/or alter the immunospecificities of the monoclonal antibodies of the library to produce and subsequently identify additional, desirable, human monoclonal antibodies. For example, the heavy (H) chain and light (L) chain immunoglobulin molecule encoding genes can be randomly mixed (shuffled) to create new HL pairs in an assembled immunoglobulin molecule. Additionally, either or both the H and L chain encoding genes can be mutagenized in a complementarity determining region (CDR) of the variable region of the immunoglobulin polypeptide, and subsequently screened for desirable immunoreaction and neutralization capabilities.
Since its conception just a few years ago, the combinatorial approach has allowed unprecedented access to the human antibody response. The cloning of antibodies from preimmune, immune, and memory compartments of the human immune system has been demonstrated. Combinatorial antibodies have been shown to provide an accurate functional reflection of the natural response as demonstrated by the ability of cloned antibodies to compete with serum antibodies for binding antigens.
Modification of the techniques that are useful in standard immunoassays can be employed for cloning unique antibodies. Immunologic assays for the detection of antigenic substances or antibodies in specimens are well known in the art. Immunoassays fall into two main categories. First, there are those immunoassays wherein one of the binding pair, either an antigen or an antibody, is attached to a solid phase, and a second binding partner, which is labeled, usually an antibody, is in the liquid phase. If the specimen contains the substance to be detected, then a complex is formed composed of the solid phase first binding partner, the substance being detected, and the detectably labeled second binding partner. In the second category are those immunoassays wherein the first and second binding partners both react freely in the liquid phase to form an immune complex if the specimen contains the substance to be detected. This immune complex is then removed from the liquid phase by binding of one of the binding partners to a carrier which is modified such that is will bind the immune complex.
An example of a sandwich immunoassay is described in David, et al., (U.S. Pat. No. 4,376,110). The assay disclosed here uses two different monoclonal antibodies from two different hybridomas. The David technique involves one of the monoclonal antibodies being bound to the solid phase throughout the assay, rather than being used in a homogeneous phage. The solid phase monoclonal antibody 1 is reacted with the liquid phase to detect the possible presence of antigen. A monoclonal antibody 2, which is labeled and soluble in liquid phase, will form, in the presence of antigen, a sandwich between solid phase monoclonal antibody 1, antigen, and labeled monoclonal antibody 2.
In Gallati, et al., United Kingdom Patent GB No. 2,074,727B, sandwich assays are described which may occur in liquid phase between (1) two monoclonal antibodies specific for two different epitopes of the same antigen, (2) a monoclonal antibody and polyclonal antibodies raised in another species to the same antigen, and (3) polyclonal antibodies raised in two different species and being directed towards different epitopes of the same antigen.
In Milich, et al., (WO 94/08597), anti-peptide antibodies are utilized as solid-phase capture reagents to bind antigen which is complexed to serum antibodies. The bound serum antibody is then detected with a labelled second antibody. In Curtis (U.S. Pat. No. 5,126,240), a first monoclonal antibody is bound to a solid matrix to form a solid support prior to formation of an admixture containing antigen. The presence of a solid phase-bound immunoreactant is then determined by the use of a second monoclonal antibody molecule.
In contrast to the present invention, standard immunoassays are utilized for the detection of the presence of antigen, antibody, or both. With its ability to provide large numbers of human antibodies directed against a single antigen, the combinatorial approach allows for the rapid assessment of immunodominant as well as neutralizing epitopes in the context of the human response. There remains a need for identifying and subsequently cloning antibodies that specifically bind to antigens known to be important targets for neutralizing antibodies, for example, even when a recombinant or purified form of the antigen is unavailable. The present invention provides a simple, high-sensitivity technique directed toward panning a population of antibody molecules, preferably in a phage display combinatorial library, in order to identify and clone antibody molecules that bind to a number of virus-neutralizing or other unknown epitopes.
The present invention provides a novel and simple xe2x80x9cantigen-capturexe2x80x9d technique to more accurately select antibody molecules, e.g., Fab, specifically binding to antigens known to be important targets for neutralizing antibodies, for example. Antibody specific for a preselected epitope, such as a non-neutralizing epitope of a desired antigen, is attached to a solid surface and used to xe2x80x9ccapturexe2x80x9d the antigen. After unbound material is washed away, a panning procedure is performed over the captured or masked antigen to identify antibodies that bind to unmasked antigens. The method of the invention is particularly useful for crude cell or viral lysates containing antigens of interest and for panning using phage display combinatorial libraries.
Thus the invention provides a method for identifying an antibody molecule that binds to an unmasked epitope on a preselected antigen, having a preselected first epitope and at least one additional epitope, comprising the steps of: a) contacting the preselected antigen having the preselected first epitope, with a first antibody molecule, under conditions which allow the first epitope of the antigen to bind to the first antibody body molecule and form an immunocomplex; b) contacting a second antibody molecule with the immunocomplex such that the second antibody molecule binds to a second, unmasked epitope on the antigen; and c) removing the second antibody molecule bound to the second epitope. Following step c), any phage, for example, expressing a second antibody molecule on its surface which binds to a second epitope, is clonally isolated and its DNA sequenced.